Carbapenem-resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae carbapenemase (KPC)- producing K. pneumoniae (KPC-Kp), have emerged as a significant hospital-acquired pathogen causing considerable morbidity and mortality in the United States and worldwide. Strikingly, a large majority of KPC-Kp isolates belong to a single sequence type, ST258. However, the molecular evolution of this epidemic clone is still very poorly understood and the genetic factors contributing to its epidemiological success remain unknown. Recent comparative genomic studies indicate that Kp ST258 strains are undergoing significant diversification in both plasmid and chromosome structures, especially at the K-antigen encoded capsular polysaccharides synthesis (cps) gene operons. Similar to what has been described in other pathogenic bacteria (e.g. Streptococcus pneumoniae), the recombination of cps-harboring region appears to be a major driving force in the genetic diversification in ST258. To date, we have found six different cps operons (cps-1 to -6) in Kp ST258 strains and identified several ST258 associated blaKPC-harboring plasmids. Notably, our preliminary data showed the variations in ST258 clades (different cps groups) are not limited to the cps regions, but also involve multiple loci that harbor genes contributing to colonization, resistance and virulence. One significant concern is that this epidemic clone may acquire enhanced virulence via chromosomal recombination and/or plasmid transfer, evolving as a highly virulent, multidrug resistant (HV-MDR) strain and further complicating current efforts to battle this crisis. Alarmingly, our preliminary results showed that certain new Kp ST258 clades (i.e. harboring novel cps operons) had higher virulence in comparison to strains from other circulating clades. Thus, we hypothesize that variable chromosomal regions and/or acquired plasmids alter host-pathogen interactions that may lead to the emergence of strains of enhanced virulence. Our objective is to identify these genomic factors and plasmid contents, and characterize their biological role associated with ST258 virulence, and examine plasmid transmissibility. In this application, we will investigate the extent to which genetic recombination has influenced genetic variation in Kp ST258. Through whole genome sequencing (WGS) of representative Kp isolates from different cps groups (cps-1 to cps-6) as well as their putative donor strains (unique STs that are likely the source of the cps replacement in ST258), we will systematically evaluate both large and small scale genomic variations in this epidemic clone and elucidate their molecular evolution history (Aim 1). Next, by using in vitro and in vivo models, we will investigate whether recombination that results in replacement of capsule-encoding machinery as well as other biologically important loci results in the emergence of HV-MDR clades (Aim 2a). In addition, we will test the hypothesis that ST258 genetic background has higher transfer efficiency and stability for KPC plasmids, which may partly explain the success of this KPC epidemic clone (Aim 2b). The results generated in this study will yield a wealth of information regarding the content and characteristics of Kp genomic variations and will likely identify novel virulence factors. The identification of genomic variations that impact the disease-causing potential of Kp strains will provide potential diagnostic and therapeutic solutions.